Kinetics of force generation by single kinesin molecules activated by laser photolysis of caged ATP

Abstract

To relate transients of force by single kinesin molecules with the elementary steps of the ATPase cycle, we measured the time to force generation by kinesin after photorelease of ATP from caged ATP. Kinesin-coated beads were trapped by an infrared laser and brought onto microtubules fixed to a coverslip. Tension was applied to a kinesinmicrotubule rigor complex using the optical trap, and ATP was released by f lash photolysis of caged ATP with a UV laser. Kinesin started to generate force and move stepwise with a step size of 8 nm at average times of 31, 45, and 79 ms after photorelease of 450, 90, and 18 M ATP, respectively. The kinetics of force generation were consistent with a two-step reaction: ATP binding, with an apparent second-order rate constant of 0.7 M ؊1 ⅐s ؊1 , followed by force generation at 45 s ؊1 per kinesin molecule. The transient rate of force generation was close to the rate of the ATPase cycle in solution, suggesting that the rate-limiting step of ATPase cycle is involved with the force generation.Motor proteins, such as kinesin, myosin, dynein, and RNA polymerase, convert the chemical energy of ATP hydrolysis into mechanical work. To investigate the mechanism of energy transduction of motor proteins, the elementary mechanical steps like force generation and sliding should be related to chemical reactions such as ATP binding, and P i and ADP release. The biochemistry of ATP hydrolysis by kinesin in solution has been investigated using native and recombinant proteins, with or without microtubules. The ATPase rate of kinesin is very low (Ͻ0.01 s Ϫ1 ) in the absence of microtubules at room temperature and is activated more than 2,000-fold by microtubules to Ͼ20 s Ϫ1 (1-5). The intermediate states of the kinesin-microtubule complex are similar to those of actomyosin, that is, M⅐K, M⅐K⅐ATP, M⅐K⅐ADP⅐P i , and M⅐K⅐ADP, where M, K, and P i are microtubule, kinesin, and inorganic phosphate, respectively. The rate-limiting step of the ATPase cycle is ADP release in the absence of microtubules, and P i or ADP release in the presence of microtubules (1-5).These biochemical results cannot be correlated directly to the mechanical cycle of kinesin, because the movements are not detected in solution. A powerful method for the investigation of mechanochemical coupling is to detect the mechanical change after laser photolysis of caged compounds, e.g., caged ATP, caged ADP, and caged P i (6, 7). Caged compounds photolyze within several milliseconds after a pulse of ultraviolet light. The mechanical reactions of actomyosin in muscle have been detected after release of ATP by photolysis of caged ATP. The rate of actomyosin dissociation after ATP release and rate of force generation were determined (6).In the experimental systems containing many molecules, individual events are averaged and the state of each molecule is not determined. Analysis of the events produced by single molecules reveals the molecular functions more directly. The kinetics of single ion channels were elucidated by stochastic analys...

Dynamic photo-control of kinesin on a photoisomerizable monolayer – hydrolysis rate of ATP and motility of microtubules depending on the terminal group

“…On further irradiation with >500 nm, the inorganic phosphate release rate reduced to 5.14×10 -3 nmol min -1 μl -1 and the difference in the release rates between E and Z isomer was 12 %. 25 Similarly, the rate of inorganic phosphate release was switched for 1b-c and 1e monolayer surfaces, with similar behaviour as seen in the gliding velocity of MT at two different isomeric surfaces (ESI Fig. S3-S6).…”

“…25 DMF was routinely dried and/or distilled prior to use and stored over molecular sieves (4A). Column chromatography was performed with silica gel (63-210μm).…”

“…And then a motility buffer consisting of BRB80 and microtubules labelled by tetramethylrhodamine succinimidyl ester were stabilized with taxol (5-10 μm in length). After keeping this for five minutes, the unbound MTs were washed out by the assay buffer containing taxol with an added oxygen 25 scavenger system (2-mercaptoethanol: 0.14 M, glucose 20 mM, catalase: 20 µg/ml, glucose oxidase: 100 µg/ml). The flow cells (2), (4), (6), (8) 8) (PSS) at 366 nm.…”

“…Several methodologies have been used for the 40 artificial control of microtubule function which includes microlithographic tracks, 21,22 electric and magnetic fields, 23 antibody 24 and so on. When considering the biodevices in the future, photochemical energy inputs offer advantages compared to chemical energy inputs because (i) light does not generate 45 waste products; (ii) it can be switched on/off easily and rapidly; (iii) photons, besides supplying the energy to the system, can also be useful to "read" the state of the system and thus to control and monitor the operation of the machine etc.. Higuchi et al 25 used the caged ATP as photo-controlled switch, because their inactive 50 caged states (OFF state of motility) can be converted to active uncaged states (ON state of motility) by UV light irradiation. Another group successfully used the photolysis of caged ATP to develop molecular shuttles on engineered kinesin tracks.…”

“…[10][11][12] The fundamental role of kinesin is to actively distribute the intracellular materials like organelles, vesicles, chromosomes along the microtubules 13,14 and to drive the cell division process 20 by converting the chemical energy of adenosine triphosphate (ATP) into mechanical energy with the positional movement towards the fast polymerizing plus ends of microtubules. [15][16][17] Each kinesin molecule has two "heads", a 50 nm long semiflexible coiled-coil region which binds to microtubules and 25 hydrolyses ATP and a "tail", which is thought to bind to the cargo. 18 Among the molecular motors, the kinesin-microtubule system is used widely for developing nanoscale biodevices, because the motility can be regenerated on a surface comparatively easily.…”